Schmidt R C, Müller A, Hain R, Bartling D, Weiler E W
Lehrstuhl für Pflanzenphysiologie, Ruhr-Universität, Bochum, Germany.
Plant J. 1996 May;9(5):683-91. doi: 10.1046/j.1365-313x.1996.9050683.x.
Nitrilase (E.C. 3.5.5.1) cloned from Arabidopsis thaliana converts indole-3-acetonitrile to the plant growth hormone, indole-3-acetic acid in vitro. To probe the capacity of ths enzyme under physiological conditions in vivo, the cDNA PM255, encoding nitrilase II, was stably integrated into the genome of Nicotiana tabacum by direct protoplast transformation under the control of the CaMV-35S promotor. The regenerated plants appeared phenotypically normal. Nitrilase II was expressed, based on the occurrence of its mRNA and polypeptide. The enzyme was catalytically active, when extracted from leaf tissue of transgenic plants (specific activity: 25 fkat mg(-1) protein with indole-3-acetonitrile as substrate). This level of activity was lower than that found in A. thaliana, and this was deemed essential for the in vivo analysis. Leaf tissue from the transgenic plants converted 1-[13C]-indole-3-acetonitrile to 1-[13C]-indole-3-acetic acid in vivo as determined by HPLC/GC-MS analysis. Untransformed tobacco was unable to catalyze this reaction. When transgenic seeds were grown on medium in the absence of indole-3-acetonitrile, germination and seedling growth appeared normal. In the presence of micromolar levels of exogenous indole-3-acetonitrile, a strong auxin-overproducing phenotype developed resulting in increased lateral root formation (at 10 microM indole-3-acetonitrile). Collectively, these data prove the ability of nitrilase II to convert low micromolar levels of indole-3-acetonitrile to indole-3-acetic acid in vivo, even when expressed at subphysiological levels thereby conferring a high-auxin phenotype upon transgenic plants. Thus, the Al thaliana nitrilase activity, which exceeds that of the transgenic plants, would be sufficient to meet the requirements for auxin biosynthesis in vivo.
从拟南芥中克隆出的腈水解酶（E.C. 3.5.5.1）可在体外将吲哚 - 3 - 乙腈转化为植物生长激素吲哚 - 3 - 乙酸。为了探究该酶在体内生理条件下的能力，编码腈水解酶II的cDNA PM255在CaMV - 35S启动子的控制下，通过直接原生质体转化稳定整合到烟草基因组中。再生植株在表型上看起来正常。基于其mRNA和多肽的出现，表明腈水解酶II得到了表达。当从转基因植物的叶片组织中提取该酶时，它具有催化活性（以吲哚 - 3 - 乙腈为底物时，比活性为25 fkat mg(-1)蛋白质）。这种活性水平低于在拟南芥中发现的水平，而这被认为对体内分析至关重要。通过HPLC/GC - MS分析确定，转基因植物的叶片组织在体内可将1 - [13C] - 吲哚 - 3 - 乙腈转化为1 - [13C] - 吲哚 - 3 - 乙酸。未转化的烟草无法催化此反应。当转基因种子在不含吲哚 - 3 - 乙腈的培养基上生长时，发芽和幼苗生长看起来正常。在存在微摩尔水平的外源吲哚 - 3 - 乙腈时，会出现强烈的生长素过量产生表型，导致侧根形成增加（在10 microM吲哚 - 3 - 乙腈时）。总体而言，这些数据证明了腈水解酶II在体内能够将低微摩尔水平的吲哚 - 3 - 乙腈转化为吲哚 - 3 - 乙酸，即使在低于生理水平表达时，也能赋予转基因植物高生长素表型。因此，拟南芥中超过转基因植物的腈水解酶活性足以满足体内生长素生物合成的需求。
